Avidin derivatives and uses thereof

ABSTRACT

A covalent conjugate of a 4′-hydroxyazobenzene-2-carboxylic acid derivative (HABA) and an avidin-type molecule, of the formula: 
                         
wherein A is (CH 2 ) n  or —CH═CH—, wherein n is an integer from 0–10; B is (CH 2 ) n  wherein n is an integer from 2 to 10; m is zero or 1; and Av is the residue of an avidin-type molecule selected from the group comprising native egg-white avidin, recombinant avidin, deglycosylated avidins, bacterial streptavidin, recombinant streptavidin, truncated streptavidin and other derivatives of said avidin-type molecules. These HABAylated avidins are red colored in the quinone configuration and can be used in many applications in the avidin-biotin technology.

This is a division of copending parent application Ser. No. 09/831,499,filed Aug. 7, 2001, now U.S. Pat. No. 6,632,929 as a national stageapplication of international application PCT/IL99/00605, filed Nov. 10,1999.

FIELD OF THE INVENTION

The present invention relates to red colored covalent conjugates of4′-hydroxyazobenzene-2-carboxylic acid derivatives (hereinafter HABA)and an avidin-type molecule, these conjugates referred herein as“HABAylated avidins”, and their application in the avidin-biotintechnology.

BACKGROUND OF THE INVENTION

Avidin and streptavidin are tetrameric proteins that, due to theirstrong interaction with biotin, have become widely useful as extremelyversatile, general mediators in a broad variety of bioanalyticalapplications, including chromatography, cytochemistry, cell cytometry,diagnostics, immunoassays and biosensing, gene probes and drug delivery(Wilchek and Bayer, 1990). The reason for their popularity is based onthe basic principle of the avidin-biotin technology, namely, that theinteraction between avidin and biotin is not affected when biotin iscovalently bound to macromolecules or to insoluble carriers (matrices).The biotin moiety can be recognized by the native avidin molecule orderivatized avidin, which contains desired reporter groups.

Avidin (from egg-white) and streptavidin (from Streptomyces avidinii)are two related proteins that bind biotin with similar dissociationconstants of about 10⁻¹⁵M. In addition to the binding of biotin, many oftheir physical properties are quite similar. Both, for example, areconstructed of four non-covalently attached identical subunits, each ofwhich bears a single biotin-binding site. The subunit M_(r) values arealso very similar. Moreover, several short stretches in the sequences ofthe two proteins are preserved, particularly two Trp-Lys stretches thatoccur at approximately similar positions.

Despite these similarities, several differences exist between the twoproteins. Avidin is a disulphide-bridged glycoprotein containing twomethionine residues, whereas streptavidin is not glycosylated and isdevoid of sulphur-containing amino acid side chains. Another significantdifference is in the tyrosine content. Avidin has only one tyrosineresidue (Tyr-33), whereas streptavidin has six tyrosine residues atpositions 22, 43, 54, 60, 83 and 96. Interestingly, the single tyrosineresidue of avidin is located in a region which contains a sequenceidentical with that of one of the streptavidin tyrosine residues (Tyr-43in the stretch Thr-Gly-Thr-Tyr).

Each avidin monomer binds one molecule of biotin. The unique feature ofthis binding, of course, is the strength and specificity of formation ofthe avidin-biotin complex. The resultant affinity constant, estimated at1.6×10¹⁵ M⁻for avidin and 2.5×10¹³ M⁻¹for streptavidin, is the highestknown for a protein and an organic ligand. It is so strong that biotincannot be released from the binding site, even when subjected to avariety of drastic conditions such as high concentrations of denaturingagents at room temperature, e.g., 6 M guanidinium hydrochloride, 3 Mguanidinium thiocyanate, 8 M urea, 10% β-mercaptoethanol or 10% sodiumdodecyl sulfate. Under combined treatment with guanidinium hydrochlorideat low pH (1.5) or upon heating (>70° C.) in the presence of denaturingagents or detergents, the protein is denatured, and biotin is dislodgedfrom the disrupted binding site.

Avidin recognizes biotin mainly at the ureido (urea-like) ring of themolecule. The interaction between the binding site of avidin with thesulfur-containing ring of the valeric acid side chain of the vitamin isof much lower strength. The relatively weak interaction between thecarboxy-containing side chain of biotin and avidin means that the formercan be modified chemically and attached to a wide variety ofbiologically active material; the biotin moiety of the resultantderivative or conjugate is still available for interaction with avidin.In turn, the avidin can be derivatized with many other molecules,notably “probes” or reporter groups of different types.

This is the crux of avidin-biotin technology (Wilchek and Bayer, 1990).Thus, a biologically active target molecule in an experimental systemcan be “labeled” with its biotinylated counterpart (a binder), and theproduct can then be subjected to interaction with avidin, eitherderivatized or conjugated with an appropriate probe.

The use of the egg-white avidin in the avidin-biotin technology issometimes restricted due to the high basicity (pI 10.5) and presence ofsugar moieties on the avidin molecule, which may lead to nonspecific orotherwise undesired reactions. In recent years, the bacterial protein,streptavidin, has largely replaced egg-white avidin for mostapplications in avidin-biotin technology. However, the problems withstreptavidin High cost and biotin-independent cell binding) haveprompted renewed interest in egg-white avidin as the standard foravidin-biotin technology. For this purpose, modified avidins exhibitingimproved molecular characteristics both over the native protein (andprevious derivatives thereof) as well as over streptavidin, have beenprepared, such as N-acyl avidins, e.g., N-formyl, N-acetyl andN-succinyl avidins. These derivatives of avidin reduce the charge of theprotein, but they are all prepared via covalent attachment to theavailable lysines of avidin, and the consequent blocking of the freeamino groups hinders subsequent preparation of other types of conjugates(notably protein-protein conjugates such as avidin-labeled enzymes)which are often prepared by crosslinking via lysine residues usingbifunctional reagents (e.g. glutaraldehyde).

A more useful and effective alternative to lysine modification is themodification via arginines. In this case, the pI of the protein isefficiently reduced and the lysines are still available for subsequentinteraction. Two different derivatives of avidin which are modified inthis manner are commercially available. One, ExtrAvidin®, can beobtained in various functionally derivatized or conjugated forms fromSigma Chemical Company (St. Louis, Mo.). A second , NeutraLite Avidin™(a product of Belovo Chemicals, Bastogne, Belgium) is additionallymodified and can be purchased in bulk quantities.

Although the reduction of the pI of egg-white avidin solves one of theproblems, the presence of the oligosaccharide residue remains a serioussource of nonspecific (biotin-independent) interaction which restrictsits application. The return of egg-white avidin as the standard foravidin-biotin technology has been contingent upon the removal of itssugars. The possibilities for removing a sugar from a glycoprotein arequite limited; it is possible to do so either chemically orenzymatically. The chemical methods currently available, e.g., using HFor periodate oxidation, are either destructive or inefficient. The wellknown enzymatic method, which employs N-glycanase, is usually veryexpensive and not very effective for avidin when conventionalmethodology is used. Eventually, a viable procedure for deglycosylationwas established and the resultant product was subsequently modifiedchemically via the arginines and is known under the trade markNeutraLite Avidin™ (Belovo Chemicals).

In spite of all these improvements, one of the main problems in theseveral applications of the avidin-biotin technology is the lack of anappropriate labeled avidin to permit the follow up of the binding ofavidin to biotinylated compounds.

In addition to its interaction with biotin, avidin is known to associatenon-covalently also with 4′-hydroxyazobenzene-2-carboxylic acid at thesame biotin-binding site of the protein, but with a lower affinity(˜10⁻⁵–10⁻⁶ M) (Green, 1965). This non-covalent association isaccompanied by a change in color from yellow to red (350 nm to 500 nm),thus allowing determination of avidin and its free binding sites.

Derivatives of 4′-hydroxyazobenzene-2-carboxylic acid and conjugatesthereof with oligo and macromolecular carriers (HABAylated molecules)are the subject of copending application of Applicants filed at the samedate as the present application.

SUMMARY OF THE INVENTION

It has now been found in accordance with the present invention thatcertain HABA derivatives covalently bound to avidins, preferably at thebinding site, form red colored HABAylated avidins (red avidins) thatchange the red color to yellow upon binding biotin. The displacement ofthe HABA moiety out of the binding site by biotin is due to a higheraffinity of biotin to the red colored avidin.

The present invention thus relates, in one aspect, to covalentconjugates of 4′-hydroxyazobenzene-2-carboxylic acid derivatives(hereinafter HABA) and an avidin-type molecule, these conjugatesreferred herein as “HABAylated avidins”, of the formula:

wherein

A is (CH₂)_(n) or —CH═CH—, wherein n is an integer from 0–10;

B is (CH₂)_(n) wherein n is an integer from 2 to 10;

m is zero or 1; and

Av is the residue of an avidin-type molecule selected from the groupcomprising native egg-white avidin, recombinant avidin, deglycosylatedavidins, bacterial streptavidin, recombinant streptavidin, truncatedstreptavidin and other derivatives of said avidin-type molecules.

When the HABA moiety is inside the avidin binding pocket, it has thequinone conformation, the conjugate has a red color and λ_(max)=504 nm:this is the HABAylated red avidin. When the HABA moiety is expelled fromthe avidin binding pocket by biotin, it has the azo configuration, theconjugate has a yellow color and λ_(max)=356 nm: this is the HABAylatedyellow avidin (see Appendix A). In the specification and claims herein,the term “HABAylated avidin” comprises both the azo and the quinoneconformations.

In the HABAylated avidins according to the invention, A is preferably—CH₂—CH₂— or —CH═CH—, and B is preferably (CH₂)₂, (CH₂)₅ or (CH₂)₆.

In the HABAylated avidins of the invention are prepared by reaction ofan avidin-type molecule with a succinimidyl ester or carbamate of HABAderivatives of formulas I and II, respectively, or a cyclic derivativeof formula III,

wherein A is (CH₂)_(n) or —CH═CH—, wherein n is an integer from 0–10;and B is (CH₂)_(n) wherein n is an integer from 2 to 10.

The HABA compounds of formulas I and II are the subject of copending PCTapplication of Applicants filed at the same date as the present PCTapplication. The cyclic derivatives of formula III are encompassed bythe present invention.

The invention further relates to columns containing immobilizedHABAylated avidins, attached to a solid support or matrix.

In another aspect, the invention relates to a single-layer proteinsystem comprising:

(i) a protein:

(ii) two ligands I and II which bind with different affinities at thesame binding site of said protein, said ligand I being the low affinityligand and said ligand II being the high affinity ligand: and

(iii) a molecule that recognizes the low affinity ligand I,

wherein in said single-layer protein system the high affinity ligand IIis buried within the binding site of the protein (i) and the lowaffinity ligand I is covalently bound to the protein and associated withthe molecule (iii) that recognizes it.

In one embodiment of the single-layer protein system aspect, themolecule (iii) may be labeled with high affinity ligand II.

In another aspect, the invention relates to a multilayer protein systemcomprising two or more layers of the single-layer protein system. In thelast layer of the multilayer protein system, the molecule (iii) ispreferably not labeled with high affinity ligand II.

In one preferred embodiment, the protein (i) in the single-layer ormultilayer protein systems is an avidin-type molecule selected from thegroup comprising native egg-white avidin, recombinant avidin,deglycosylated avidins, bacterial streptavidin, recombinantstreptavidin, truncated streptavidin and other derivatives of saidavidin-type molecules; the low affinity ligand I is HABA(4′-hydroxyazobenzene-2-carboxylic acid) or a HABA derivative, the highaffinity ligand II is biotin, and the molecule (iii) that recognizesligand I is an anti-HABA antibody or a biotinylated anti-HABA antibody.

The anti-HABA antibody used according to the invention may be polyclonalor monoclonal, and can be prepared by immunization of rabbits and mice,respectively, with a conjugate of HABA and an immunogenic protein, suchas for example HABA-KLH. The anti-HABA antibodies are the subject ofcopending application of Applicants filed at the same date as thepresent application.

In another embodiment, the protein (i) in the single-layer or multilayerprotein system is anti-dinitrophenyl (DNP)-antibody; the low affinityligand I is trinitrobenzene (TNP) or mononitrobenzene (MNP), the highaffinity ligand II is DNP and the molecule (iii) that recognizes ligandI is a MNP- or TNP-tagged anti-DNP antibody.

The single-layer or multilayer protein system according to the inventionmay be formed on a substrate such as gold, silicium, polystyrene.Preferably, the multilayer protein system will comprise 5–6 layers.

In another aspect, it is provided a method for assembling a single-layerprotein system according to the invention, which comprises the steps of:

(a) covalently binding said low affinity ligand I to said protein (i),thus obtaining a low affinity ligand I-protein (i) complex in which saidligand I is buried within the binding site of said protein (i) and isthus not available for interaction with other molecules that recognizeit;

(b) reacting the high affinity ligand II or a compound containing saidhigh affinity ligand II with the low affinity ligand I-protein (i)complex of step (a) above, whereby low affinity ligand I is expelledfrom within the binding site to the periphery but remains covalentlybound to protein (i) and high affinity ligand II is associated to, andburied within, the binding site of protein (i); and

(c) reacting the low affinity ligand I-protein(i)-high affinity ligandII complex of step (b) with a molecule (iii) that recognizes and bindsto low affinity ligand I and can be labeled with high affinity ligandII.

In still another aspect, it is provided a method for assembling amultilayer protein system according to claim 3, which comprises thesteps of:

(a) covalently binding said low affinity ligand I to said protein (i),thus obtaining a low affinity ligand I-protein (i) complex in which saidligand I is buried within the binding site of said protein (i) and isthus not available for interaction with other molecules that recognizeit;

(b) reacting the high affinity ligand I or a compound containing saidhigh affinity ligand II with the low affinity ligand I-protein (i)complex of step (a) above, whereby low affinity ligand I is expelledfrom within the binding site to the periphery but remains covalentlybound to protein (i) and high affinity ligand II is associated to, andburied within, the binding site of protein (i);

(c) reacting the low affinity ligand I-protein(i)-high affinity ligandII complex of step (b) with a molecule (iii) that recognizes and bindsto low affinity ligand I and is labeled with high affinity ligand II;and

(d) reacting the protein system of step (c) with low affinity ligandI-protein (i) complex as in step (b) above, and repeating steps (c) and(d) as desired.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the signaling system according to theinvention. HABAylated red avidin (

), λ_(max)=504 nm (see red spectrum on the right side) does not interactwith the anti-HABA antibody (

). The cascade is triggered upon addition of biotin (B) orbiotin-containing molecules, which expel the covalently attached HABAmoiety (H) from the binding site. The spectrum shows a shift to yellow,λ_(max)=356 nm. The HABA group is now available for subsequentinteraction with anti-HABA antibody or avidin. Reaction with avidinrestores the red color.

FIG. 2 illustrates the reaction of avidin with the cyclicHABA-derivative (Compound 11) in the presence (x) or absence ( ) ofbiotin. Avidin was incubated with an equimolar (per subunit)concentration of the cyclic HABA in aqueous buffer at pH 8, and thechange in absorbance at 500 and 330 nm was followedspectrophotometrically.

FIG. 3 shows the results of ELISA assay using the polyclonalaffinity-purified anti-HABA antibody. Plates were coated with HABAylatedavidin, the desired dilutions of antibodies were applied with biotin (♦Antibody purified on column A,

Antibody purified on column B) or without biotin (⋄ Antibody purified oncolumn A,

Antibody purified on column B) and the plates were assayed using asecondary antibody-enzyme conjugate.

FIG. 4 illustrates an artificial cascade formed by layers generated byconsecutive addition of HABAylated avidin and biotinylated antibodies.ELISA plates were coated with biotinylated-BSA

to which HABAylated avidin (other symbols are as in FIG. 1) andbiotinylated anti-HABA antibody was added. The cycle was continuedseveral times. Formation of the layers was detected using a secondaryantibody-enzyme conjugate. The signal of the first layer was set asbinding index=1.

DETAILED DESCRIPTION OF THE INVENTION

The term “avidin-type molecule” as used herein refers to the nativeegg-white glycoprotein avidin, to deglycosylated forms of avidin, tobacterial streptavidins produced by selected strains of Streptomyces,e.g., Streptomyces avidinii, to truncated streptavidins, and torecombinant avidin and streptavidin as well as to derivatives of native,deglycosylated and recombinant avidin and of native, recombinant andtruncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl,N-phthalyl and N-succinyl avidin, and the commercial products ExtrAvidinand Neutralite Avidin.

All forms of avidin-type molecules are encompassed by the presentinvention, both native and recombinant avidin and streptavidin as wellas derivatized molecules, e.g. nonglycosylated avidins, N-acyl avidinsand truncated streptavidins. Some of these materials are commerciallyavailable, e.g. native avidin and streptavidin, nonglycosylated avidins,N-acyl avidins and truncated streptavidin, or can be prepared bywell-known methods (see Green, 1990, for preparation of avidin andstreptavidin; Hiller et al., 1990, for preparation of non-glycosylatedavidin; Bayer et al., 1990, for the preparation of streptavidin andtruncated streptavidin). Recombinant avidin and streptavidin can beprepared by standard recombinant DNA techniques, for example, asdescribed by Chandra and Gray, 1990, for recombinant avidin, and byArgarana et al., 1986, for recombinant streptavidin.

The “biotinylated ligands” that can be used with the modified avidins ofthe invention in methods of application of the avidin-biotin technology,are biotinylated forms of desired ligands such as proteins, e.g.antibodies, enzymes, lectins, or carbohydrates and glyco-conjugates,e.g. glycoproteins, gangliosides, heparin, polysaccharides, or nucleicacids, i.e. DNA and RNA, or phages, viruses, bacteria and other cells,wherein said ligands are covalently linked to biotin or to a homologue,analog or derivative thereof. Many biotinylated ligands are commerciallyavailable or can be prepared by standard methods (see, for example,Bayer and Wilchek, 1992).

HABAylated avidins as described in the invention can be used as verysensitive tools for the recognition and visualization of biotin andbiotinylated molecules. They can replace avidin in all the applicationsof the avidin-biotin technology where avidin-like molecules arecurrently used (molecular and cell biology, biochemistry anddiagnostics) with the main advantage of providing a strong signalenhancement with minimum background.

The concept behind the use of this new colored avidin is the fact that,when biotin or biotin-containing molecules are added, the HABA moieties,while remaining covalently linked to the avidin molecule, are expelledfrom the binding pocket and remain available at the avidin surface.After binding of biotin, the HABA moieties are used as tags for thefollowing addition of anti-HABA antibodies (labeled or non-labeled).Both the HABAylated avidin and/or the anti-HABA antibodies can belabeled with biotin or its derivatives, different chromophores orenzymes, thus allowing visualization of the complex formed. Biotin orbiotin-containing molecules can be used to expel only parts of the HABAfrom the red avidin, leaving the other HABA residues still buried in thebinding site. It is important to note that, upon biotin binding to theHABAylated avidin, the HABA molecules shift from the quinone to the azoconformation (see Appendix A) and a color change from red to yellow isobserved.

The invention also relates to a HABAylated avidin-type molecule of theinvention attached to a solid support or matrix. Any solid support usedin the art is suitable such as, but not limited to, resins, microtiterplates, glass beads, magnetic beads and the like. The attachment of theHABAylated avidin to the solid support may be covalent or noncovalentand is carried out by standard methods. In one preferred embodiment, theHABAylated avidin-type molecule is immobilized onto a resin, preferablySepharose, and the thus obtained Sepharose-HABAylated-avidin affinityresin may be poured into a column for isolation procedures (Bayer andWilchek, 1992). In the description herein the term “avidin-Sepharosecolumn” will be used for a column that contains a HABAylated avidin-typemolecule of the invention immobilized onto a Sepharose resin. Thesecolumns are useful particularly for separation procedures.

In another embodiment of the invention, the HABAylated avidin-typemolecule is attached to wells of microtiter plates.

The red HABAylated avidins of the invention can be used for thedetermination of biotinylated sites on proteins. They also permit tofollow visually the attachment of avidin to biotinylated molecules,which has not been possible as yet. The binding of the HABAylated avidinto biotin-containing molecules can also be monitored with anti-HABAantibodies, both monoclonal and polyclonal, thus providing an additionalmeans for detection and a convenient tool for application using theavidin-biotin system.

The anti-HABA antibodies are the subject of a copending applicationfiled on the same date and are of two categories: antibodies thatrecognize the HABA moiety on any protein including avidin, and thosethat recognize the HABA moiety on avidin only after the addition ofbiotin or biotinylated protein (i.e., after exposing the HABA moiety).However, the second type of antibody can develop the binding cascade ina very specific and ordered way. The first type of antibody, being lessspecific, can recognize HABA when it is either inside or outside theavidin pocket and it does not allow such a delicate control over theamplification cascade.

According to the present invention, it could be determined that proteincascades or multilayers of proteins can be formed artificially, wherebythe binding of one molecule depends on the signaling of another. Such asystem can be achieved using two molecules which display differingaffinities for the same binding site of a protein, such as biotin andHABA for avidin, or dinitrobenzene and tri-(or mono)nitrobenzene foranti-dinitrophenyl antibody. If the lower-affinity ligand (e.g. HABA ortri-(or mono)nitrobenzene) is coupled covalently to the binding site,the high-affinity ligand (e.g. biotin or dinitrobenzene) can be used todisplace it from the binding pocket. The expelled moiety, stillcovalently attached to the protein, is now available for furtherinteraction with other molecules that can bind to this low-affinityligand (e.g. optionally biotinylated anti-HABA antibody or optionallyMNP- or TNP-tagged anti-DNP antibodies). Consequently, one interactionwill be dependent on the previous one, thus enabling to trigger acascade of binding, i.e., to construct an organized system of proteinmultilayers, thereby increasing the signal tremendously.

The possibility of building organized protein layers only inbiotinylated sites of a surface can be a great advantage in manybiophysical applications such as in protein chips, biosensors, etc.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES

List of Compounds

In the Examples, the following compounds 1–11, which formulas arepresented in Appendix B hereinafter Gust before the Claims), will beidentified by their numbers in bold. Compounds 8, 10 and 11 are used asstarting materials for the preparation of the HABAylated avidins.Compounds 11 are part of the present invention.

-   -   0. 4′-hydroxyazobenzene-2-carboxylic acid (HABA)    -   1. 3-(2-hydroxyphenyl)propionic acid A=(CH₂)₂    -   2. N-succinimidyl 3-(2-hydroxyphenyl)propionate A=(CH₂)₂    -   3. N-6-(t-butoxycarbonylamino)hexyl        3-(2-hydroxyphenyl)propionamide A=(CH₂)₂; B=(CH₂)₆    -   4. N-6-(methoxycarbonyl)pentyl 3-(2-hydroxyphenyl)propionamide        A=(CH₂)₂; B=(CH₂)₅    -   5. N-5-carboxypentyl 3-(2-hydroxyphenyl)propionamide A=(CH₂)₂;        B=(CH₂)₅    -   6.        3′-(6-t-butoxycarbonylamino)hexylaminocarbonylethyl4′-hydroxy-azobenzene-2-carboxylic        acid A=(CH₂)₂; B=(CH₂)₆    -   7.        3′-(6-aminohexylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylic        acid A=(CH₂)₂; B=(CH₂)₆    -   8.        3′-(6-(succinimidyloxycarbonylamino)hexylaminocarbonylethyl)-4′-hydroxy-azo-benzene-2-carboxylic        acid A=(CH₂)₂; B=(CH₂)₆    -   9.        3′-(5-carboxypentylaminocarbonylethyl)-4′-hydroxy-azobenzene-2-carboxylic        acid A=(CH₂)₂; B=(CH₂)₅    -   10.        3′-(5-succinimidyloxycarbonylpentylaminocarbonylethyl)4′-hydroxyazobenzene-2-carboxylic        acid A=(CH₂)₂; B=(CH₂)₅    -   11. cyclic HABAs        ABBREVIATIONS: BCA: bicinchoinic acid; BOC: t-butoxycarbonyl;        BSA: bovine serum albumin; DCC: N,N′-dicyclohexylcarbodiimide;        DG-avidin: deglycosilated avidin; DMAP: dimethylaminopyridine;        DMF: N,N′dimethyl formamide; DSC: disuccinimidylcarbonate; HABA:        4′-hydroxyazobenzene-2-carboxylic acid; KLH: Keyhole Lympet        Hemocyanin; NHS: N-hydroxysuccinimide; Su: succinimidyl; TEA:        triethylamine; TPCK:        L-1-Tosylamide-2-phenylethylchloromethylketone; TSTU:        tetramethyluronium tetrafluoroborate.        Materials and Methods

-   (i) Materials. Triethylamine (TEA) and    NN′-dicyclohexylcarbodiimide (DCC) were obtained from Merck    (Darmstadt, Germany), N-BOC-1,6-diaminohexane and TSTU were obtained    from FLUKA, (Buchs, Switzerland), disuccinimidylcarbonate (DSC) was    purchased from Calbiochem (La Jolla, Calif. USA);    3(2-hydroxyphenyl)propionic acid and anhydrous hydrochloric acid    solution in dioxane were purchased from Aldrich (Milwaukee Wis.,    USA). Egg-white avidin was provided by STC laboratories (Winnipeg,    Canada); deglycosilated avidin (DG-avidin) by Belovo (Bastogne,    Belgium) and streptavidin by Boehringer Mannheim (Mannheim,    Germany). DG-avidin was prepared according to the procedure of    Hiller, 1990. N-hydroxysuccinimide, biotin, HABA, BSA, TCPK treated    trypsin, Keyhole Lympet Hemocyanin (KLH) and all the other chemicals    were obtained from Sigma Chemicals (St.Louis, Mo., USA). Sepharose    CL4B and Sephadex G25 was purchased from Pharmacia Biotech AB    (Uppsala, Sweden) Peroxidase-conjugated anti-rabbit IgGs were    obtained from Jackson ImmunoResearch (West Grove, Pa., USA). UV    spectra were recorded with a Milton Roy spectronic UV-Vis    spectrophotometer, mod. 1201; HPLC analysis was carried out on a    Vidac ‘Protein and Peptides C₁₈′ column, using a Waters pumping    system 600E, a Knaur variable wavelength detector and a Hewlet    Packard model 3390A integrator.

-   (ii) Biotinylation Procedures. The proteins and enzymes used in the    Examples were biotinylated by conventional biotinylating methods    using biotinyl N-hydroxysuccinimide ester (BNHS) as described    previously (Bayer and Wilchek, 1990).

-   (iii) Enzyme assays. Horseradish peroxidase activity (HSP).    Peroxidase activity was determined using o-phenylenediamine (oPD) as    substrate. Substrate solution included 8 mg of the substrate per 20    ml in citrate-phosphate buffer (50 mM), pH 5, to which 10 μl of 30%    hydrogen peroxide was added. The reaction was stopped using 1M    H₂SO₄. Color formation was measured at 490 nm.

-   (iv) Protein. Protein was determined by the Bradford method using    either avidin or streptavidin (where appropriate) or BSA as a    standard. HABAylated protein was determined using the BCA assay    (Pierce, Rockford, Ill., USA).

EXAMPLE 1

Synthesis of Compound 10

1.1 Synthesis of Compound 4

To a solution of Compound 1 (0.997 g, 6 mmol) in CH₂Cl₂ (25 ml) wereadded ε -aminocaproic acid methyl ester (1.74 g, 12 mmol), an equimolaramount of TEA (1.6 ml, 12 mmol) and DCC (1.36 g, 6.6 mmol). The reactionwas carried out for 4 hours in an ice bath. The solution was washedthoroughly with water, HCl (0.05 M), water, bicarbonate (0.1 M) andagain with water. The CH₂Cl₂ fraction was dried over sodium sulfate andthe pure product Compound 4 was obtained by precipitation with absolutediethylether.

1.2 Synthesis of Compound 5

Compound 4 (330 mg, 1.08 mmol) was dissolved in methanol and 5.4 ml 0.5M NaOH were added thereto. After 1 hour, the reaction mixture wasbrought to pH 2 with HCl and the methanol removed by evaporation. Theoily mixture was dissolved in hot ethyl acetate and the pure productCompound 5 crystallized upon cooling down.

1.3 Synthesis of Compound 9

Compound 9 was synthesized from the Compound 5 following the sameprocedure described in Example 2.3 below for Compound 6.

1.4 Synthesis of Compound 10

Compound 10 was prepared from Compound 9 by two different procedures:

a. Activation with NHS

The synthesis was carried out using the same procedure described inExample 2.1 below for Compound 2. Compound 9, DCC/CH₂Cl₂ and NHS wereused in equimolar concentrations to avoid activation of the carboxylgroup at the second phenyl ring. The urea derivative was removed byfiltration, and Compound 10 was washed with water and dried.

b. Activation with TSTU (Bannwarth, 1991)

TSTU (70.4 mg, 0.24 mmol) and DMAP (57 mg, 0.48 mmol) were added toCompound 9 (100 mg, 0.24 mmol) dissolved in a mixture ofDMF/dioxane/water (1/1/0.5). After complete conversion (30 min),Compound 10 (purity 96%) was lyophylized and further purified by HPLC.1.5 Synthesis of analogs of Compound 10

The same synthesis was successfully carried out for a compound wherein Ais —CH═CH═ using 2-hydroxycinnamic acid as the starting compound in thefirst step instead of Compound 1. Other HABA derivatives can be obtainedfrom similar hydroxyphenyl reagents carrying different spacer arms inposition 2.

EXAMPLE 2

Synthesis of Compound 8

2.1 Synthesis of Compound 2

To a cooled solution of Compound 1 (0.997 g, 6 mmoles) in CH₂Cl₂ (21ml), NHS (0.828 g, 7.2 mmoles) and DCC (1.485 g, 7.2 mmoles) were added.After 3.5 h, the solution containing Compound 2 was filtered anddirectly used for the next synthetic step, without any furtherpurification.

2.2 Synthesis of Compound 3

N1-BOC-1,6-diaminohexane (1.52 g, 6 mmoles) was added, while stirring,to the dichloromethane solution of Compound 2, followed by 835 μl (6mmoles) of TEA. The reaction was stirred overnight at room temperature,filtered and evaporated to dryness. The product was redissolved in ethylacetate and the organic solution was washed (with diluted NaHCO₃,diluted citric acid and water), dried over Na₂SO₄ and evaporated todryness. Diethyl ether (30 ml) was added to the resulting oil, theprecipitated impurities were removed by filtration, and the solutioncontaining Compound 3 was evaporated to dryness and used further.

2.3 Synthesis of Compound 6

To cooled anthranilic acid (0.750 g, 5.45 mmoles) and NaNO₂ (0.377 g,5.45 mmoles) dissolved in water (15 ml), 1.5 ml of concentrated HCl wereadded. After 10 min, the solution was dropwise added to Compound 3 (1.62g, 5.45 mmoles) dissolved in a mixture of methanol:0.5M KOH,1:1 (15 ml).The pH was controlled and adjusted to 8.0 using HCl and KOH. After 20min, methanol was removed by evaporation, the solution was acidified topH 3–4 with diluted citric acid, and the solid product was extractedwith ethyl acetate. The organic solution was washed with water, driedover Na₂SO₄ and evaporated to dryness, thus obtaining Compound 6.

2.4 SynthesisofCompound 7

A solution of Compound 6 in dioxane was dried, filtered, and HClsaturated dioxane was added. After 1 hour, the product Compound 7precipitated as the hydrochloride salt, was isolated by filtration,washed with diethyl ether and dried.

2.5 Synthesis of Compound 8

A solution of Compound 7 (35.8 mg, 0.08 mmoles) in DMF (0.24 ml) wasslowly added into portions (8×30 ?l), while stirring, to a solution ofDSC (41 mg, 0.16 mmoles) in CH₃CN (1.6 ml). After each addition, 2equivalents of TEA (with respect to Compound 7) were also added, and thepH monitored continuously and maintained below 4.0. Five minutes afterthe last addition of the HABA-derivative 7, 2 ml of 1N HCl were added.The product Compound 8 crystallized as a fine powder. It was isolated byfiltration, washed with diluted HCl and dried.

EXAMPLE 3

Synthesis of Compound 11

The synthetic pathway leading to Compound 11, the cyclic form of HABA,via Compound 8, is represented in Scheme 2.

Compound 11 is readily obtained from Compound 8 in neutral aqueousbuffer. A concentrated DMF solution of Compound 8 (10 mg/ml) is dilutedin PBS and pH is adjusted to 8.0 with small additions of 4% NaHCO₃.Transformation of the active carbamate 8 into the cyclic HABA derivative11 is verified by TLC (CHCl₃/MeOH 20%) and UV spectrophotometry. In theconditions used, total conversion is obtained after 24 hours standing atroom temperature.

EXAMPLE 4

Preparation of HABAylated Avidins

Avidin-type molecules were affinity labeled with HABA reagents 8, 10 and11. The cyclic Compound 11 was designed such that the HABA moiety wouldremain covalently attached to the binding site of avidin. The orthoposition of the HABA hydroxy group was thus modified with a reactivefunctional group, which, due to steric constraints, forms anintramolecular cyclic carbamate (Scheme 1). This cyclic HABA 11 ishydrolyzed by avidin enabling exploitation of the principle of forcedcatalytic hydrolysis (Vetter et al., 1994) to attach the HABA moiety toan appropriate site in or near the binding site of avidin. In theconditions used, only the primary amino group directly involved in thepseudo-enzymatic reaction (Lys 111 in avidin) can be derivatized withthis reagent. The cyclization of the reagent was necessary since theactivated linear N-hydroxy-succinimidocarbamate would react with anyamino group, whereas the cyclic compound reacts in situ, i.e.,selectively, after occupying the binding site of avidin. This results ina red HABAylated avidin that is labeled with 4 HABA molecules occupyingthe 4 binding sites of avidin.

Reagents 8 and 10 are less specific. These HABA derivatives which areactivated as N-succinimidyl carbamate and N-succinimidyl ester,respectively, react with primary amino groups at the level of anyprotein, peptide or amino-carrying polymer surface. Binding occursthrough any primary amino function, therefore, they can react with anylysine on the avidin/streptavidin surface. In this case, coupling in thearea of the (strept)avidin's biotin/HABA binding pocket occurs as in aconventional labeling, where the affinity for a specific site of theprotein is directing the site of labeling. Additional labeling at theperiphery of the avidin will result in an additional peak at 357 nm.Binding of anti-HABA antibodies to HABA will be independent of biotinfor the HABA moiety outside of the binding pocket at the periphery andbiotin-dependent for the HABA bound inside the binding pocket. This HABAmust be expelled from the binding site by addition of biotin prior totheir binding to the antibodies.

4.1 HABAylation of Avidin with Compound 8

10–50 ml of a concentrated solution of Compound 8 in DMF (5–30 mg/ml)were added, while stirring, to a solution of an avidin-type molecule inaqueous buffer at pH 8.0–8.5 (2–10 mg/ml). The molar ratio between theHABA-succinimidyl carbamate derivative 8 and the avidin-type moleculewas between 50 and 400, depending on the starting avidin concentrationand the desired degree of modification. The reaction was carried out atroom temperature or lower for 1–2 hours.

4.2 HABAylation of Avidin with Compound 10

A freshly prepared solution of HABA-N-succinimidyl ester 10 (2.5 ml) inEtOH:PBS, 1:3 (1–50 mg/ml) was added, while string, to a solution of anavidin-type molecule (2–20 mg) in 1 ml of phosphate buffer, pH 7.4containing 0.5M NaCl. The molar ratio between the ester derivative andthe avidin-type molecule was between 4 and 100 depending on the startingprotein concentration and the desired degree of modification Addition ofthe HABA resulted in an immediate shift of the spectrum to 504 nm. Thereaction was carried out at room temperature for 2–3 hours. HABAylationswere carried out using egg-white avidin, streptavidin, Neutralite avidinand DG-avidin.

EXAMPLE 5

HABAylation of Avidin with Compound 11

5.1 Preparation

Compound 11, prepared from Compound 8, was added to an avidin solutionin PBS. A shift in the spectrum to 504 nm was observed that developsgradually. The same effect was observed for deglycosylated avidin,Neutralite avidin and streptavidin The reaction at room temperature wasmonitored by UV and followed by measuring either absorbance at 500 and331 nm, or the ratio between them, as parameters. When no further changein those parameters was observed, the modified avidin was purified fromexcess reagent by gel filtration. Different reaction conditions varyingeither the protein concentration (0.8–3 mg/ml) or the HABA/subunit ratio(between 1 to 10) have been used and the same final product, containingone HABA/subunit, was obtained. High protein concentration as well ashigh HABA/subunit ratios allow faster reactions. As shown in FIG. 2, noshift was observed if the cyclic reagent 11 was added together withbiotin (x), indicating that biotin occupied the binding site andprevented the reaction with HABA.

5.2 Characterization

5.2.1 Concentration of HABA-Labeled Avidin

Affinity labeling with HABA influences the absorbance of avidin in the280 nm area. Therefore, avidin molar extinction constant at 280 nm(ε^(1%) _(280 nm)=15.4) cannot be used to determine the concentration ofthe HABA-labeled avidin derivative. The most commonly used colorreagents for determination of protein concentration are also affected bythe HABA molecule. Among the methods tested, the BCA protein assay, evenif not accurate, proved to be the most reliable one. ε^(1%) _(280 nm) ofa fully HABAylated avidin with Compound 11 was calculated from a seriesof experiments as being 21.9. Molar extinction value of HABA inside thecomplex pocket was determined to be ε_(504 nm)[cm⁻¹M⁻¹]=33,000. Ifcomplete HABAylation is obtained, this value can also be used to assessthe concentration of the avidin-HABA conjugate.

5.2.2 Spectrophotometric Characterization

UV spectrum of the purified affinity HABA-labeled avidin obtained withcompound 11 was recorded. As shown in FIG. 1, the HABAylated-avidinshows a maximum at 504 nm. After addition of biotin, the HABA moiety isexpelled from the binding site resulting in a shift of color from red toyellow (λ_(max)=356 nm). Addition of avidin restores the red color andthe maximum shifts back to 504 nm.

5.2.3 Number of HABA Molecules/Avidin Subunits and Localization of theHABA

HABA-labeled avidin-type molecules carry one single HABA molecule perprotein subunit. Precise determination of the number of HABAmolecules/avidin subunit was assessed by mass spectrometry on a labeledDG-avidin sample (not shown). The analysis of the non-HABAylatedDG-avidin showed an m/z value of 14287–14290, corresponding to a singlesubunit of DG-avidin. The, HABAylated DG-avidin showed a single peak(m/z 14727–14734), the difference in mass (about 440 units) beingconsistent with the molecular weight of the HABA moiety.

To determine which amino acid residue of the DG-avidin molecule wasmodified by HABA, the HABAylated DG-avidin was subjected to trypsindigestion. The hydrolysate was separated by HPLC, and the sequence ofthe orange-colored HABAylated peptide was analyzed. The analysis showedthat the Lys-111 residue was HABA-modified.

5.2.4 Stability and Storage of the HABA-Labeled Avidins.

Avidins are known for their good stability. Similarly, the affinityHABA-labeled avidins obtained according to the invention are verystable:

PBS or neutral pH solutions of HABAylated-avidins can be stored at 4° C.for several months if kept in a sterile environment;

Complete heat denaturation of HABAylated-avidins in PBS solutionrequires more than 2 hours treatment at 100° C.

The HABAylated-avidins can be freeze-dried for long term storage eitherfrom PBS or salt free solution. Original solutions can be readilyre-obtained upon addition of the proper buffer.

Short term standing (1 hour) at high (0.1N NaOH) or low pH (0.1M aceticacid) do not damage the HABAylated-avidins.

EXAMPLE 6

Labeling of HABAylated Avidins with Non-HABA Markers

Since only one lysine per subunit is covalently modified by HABA in theaffinity HABAylated avidin (see Example 5.2.3 above), the other primaryamino functions on the protein surface are available for furthercoupling. Thus, other labels such as fluorescent, chemiluminescent, dyemolecules or enzymes can be covalently linked to the HABAylated-avidinmolecule by procedures used for general protein non-radiaoctive labeling(Garman, 1997). According to the invention, the HABAylated-avidin ofprevious examples was further labeled with FITC, a fluorescent molecule,and with the dye dinitrobenzene (DNP), Analysis of the resultingproducts showed that the FITC and DNP moieties were covalently linked toother amino groups of the HABAylated avidin molecule.

EXAMPLE 7

Anti-HABA Antibodies

7.1 Preparation of Immunogenic HABAylated Proteins

KLH, BSA, and goat-affinity purified anti-Mouse IgGs were HABAylatedusing the succinmidyl reagents (Compound 8, 10) as coupling agent by theprocedure described in Example 4.1 above for avidin. Briefly, thecoupling agent dissolved in DMF was added to a solution of the proteinin 0.1M NaHCO₃ (2–10 mg/ml). After 2 hours, the excess of HABA reagentwas removed by gel filtration on a G25 column. The degree of couplingcould be estimated from the UV spectra of the conjugates, consideringthe ε₃₅₆ of 12,900 for the HABA-derivative in PBS and measuring theprotein concentration by BCA protein assay.

7.2 Preparation of Anti-HABA Polyclonal Antibodies

Rabbits (12 weeks) were immunized by intradermal injection of 0.5 mg ofHABA-KLH (carrying ˜50 molecules of HABA/protein) emulsified in completeFreund's adjuvant Boosts were administered after 4 weeks by injecting0.5 mg of HABA-KLH in incomplete Freund's adjuvant. Blood was collectedfrom the ear vein two weeks after boosting and serum was isolated bycentrifugation and preserved at −20° C. Preserum was collected beforeimmunization and used as a control.

7.3 Affinity Purification of Anti-HABA Polygonal Antibodies

Two different Sepharose gels (A and B) were prepared for the isolationof anti-HABA antibodies specific to different epitopes in the HABAmolecule. A schematic representation of the two gels is described inScheme 2.

-   -   GEL A is a highly functionalized HABAylated-Sepharose having the        correct HABA moiety (4′-hydroxy-azophenyl-2-carboxylic acid        linked at position 3′) connected with a spacer arm in a similar        way as in the HABAylated KLH.    -   GEL B is a highly functionalized gel having a HABA moiety with a        slightly different structure (2′-hydroxy-azophenyl-2-carboxylic        acid linked at position 5′ via a spacer arm) obtained by        diazotization of tyramine-Sepharose with anthranilic acid.

These two gels allow isolation of anti-HABA antibodies with differentcharacteristics: GEL A is able to isolate anti-HABA antibodies thatrecognize the whole HABA molecule, while GEL B allows the isolation ofantibodies that are specific for the azophenyl-2-carboxylic acid moietyof the HABA core.

7.4.a Preparation of GEL A

Sepharose CL-4B hydroxyl functions were first activated as p-nitrophenylcarbonates (Wilchek et al, 1984) and the active gel was then coupled toa 2-hydroxyphenyl derivative carrying a spacer arm with a primary amineas the terminal group. Different spacers can be introduced by varyingthis first compound. The HABA function was then obtained bydiazotization of the phenyl residues directly on the Sepharose support(Vetter et al, 1994).

(i) Sepharose-tyrosine or 2-hydroxyphenylpropionyldiamino hexane.

The primary amines tyrosine or 2-hydroxyphenylpropionyl-diaminohexanedissolved in aqueous buffer (35 mM borate buffer, pH 8.5) were added tothe p-nitrophenyl carbonate-activated Sepharose (carrying 50–100 mmolesof active group/g of wet gel).

Reactions were carried out on 3–5 g of gel, in a total volume of 12–15ml and using a molar ratio of 3:1 between the primary amine and theactivated groups of the gel. Suspensions were gently stirred for 150minutes at room temperature, and the gels were then washed with water,MeOH, EtOAc and then, MeOH and water again. Unreacted active groups inthe gel were hydrolyzed by 5 minutes exposure to 0.2M NaOH. Gels werewashed again and resuspended in 0.2M KOH (3 g/5 ml) for the finaldiazotization step.

(ii) Diazotization Reaction.

Anthranilic acid and NaNO₂ were dissolved in H₂O (156 mmoles/ml forboth) and concentrated HCl (100 ml/ml of water) was added after coolingin an ice bath. The solution was stirred for 5 minutes and then addeddropwise to the gel suspended in 0.2M KOH (3 g/5 ml). The reaction wasgently stirred for 15 minutes, while the temperature was controlledusing an ice bath, and the pH was monitored constantly and adjusted to8.0–8.5 using diluted KOH. A molar ratio of 1:1 between anthranilic acidand the phenyl residues in the gel was used, assuming that a completeconversion of the activated p-nitrophenyl groups occurred in theprevious step of the synthesis. The GEL A obtained was then washed (sameprocedure as in previous step) and suspended in PBS.

7.4.b Preparation of GEL B

Hydroxyl groups of Sepharose CL-4B were first activated withN,N′-disuccinimidylcarbonate (Wilchek and Miron, 1985). and theactivated gel was then coupled to tyramine via the amino group. The HABAderivative was obtained by diazotization of the phenyl residues usinganthranilic acid

(i) Sepharose-tyramine

Tyramine dissolved in PBS (pH 7.4) was added to theN,N′-disuccinimidylcarbonate activated Sepharose (carrying 20–80 mmolesof active groups/g of wet gel). Reactions were carried out on 3–5 g ofgel, in a total volume of 12–15 ml and using a molar ratio of 5:1between the primary amine and the activated groups of the gel.Suspensions were gently stirred overnight at 4° C. Gels were then washedextensively until no more free amine could be detected and unreactedactive groups in the gel were hydrolyzed by 5 minutes exposure to 0.2MNaOH. Gels were washed again and resuspended in 0.2M borate buffer (pH8.5) (3 g/5 ml) for the final diazotization step.

(ii) Diazotization Reaction.

Anthranilic acid and NaNO₂ were dissolved in H₂O and concentrated 0.2MHCl was added after cooling in a ice bath. The solution was stirred for5 minutes and then added dropwise to the gel suspended in 0.2M boratebuffer (3 g/5 ml). The reaction was gently stirred for 15 minutes, whilethe temperature was controlled using an ice bath and the pH monitoredconstantly and adjusted to 8.0–8.5 using diluted KOH. A molar ratio of1:1 between anthranilic acid and the phenyl residues in the gel wasused, assuming that a complete conversion of the activated p-nitrophenylgroups occurred in the previous step of the synthesis. The GEL Bobtained was then washed and finally suspended in PBS.

7.5 Affinity Purification of Anti-HABA Polyclonal Antibodies with GEL Aand GEL B.

Sepharose GELS A and B were pre-treated with 0.1M TEA pH 11.5 before anyfurther use and re-equilibrated with PBS. Rabbits' antisera diluted 1:1with PBS or IgG antibodies obtained by (NH₄)₂SO₄ precipitation wereincubated with the gel for 4 hours at 4° C. Total removal of anti-HABAantibodies from supernatant was verified by dot blot on nitrocellulosepaper, using BSA-HABA for dotting. In order to obtain an efficientretention of anti-HABA antibodies, a ratio of 1:1 w/w between serum andgel was used. The gel was then washed extensively with 0.05 M Tris HCl,0.5M NaCl, pH 7.5. Bound antibodies were finally eluted by basictreatment, using 0.1M TEA, pH 11.5, immediately neutralized and dialyzedagainst PBS.

7.6 Characterization of Anti-Sera and Affinity Purified Anti-HABAPolyclonal Antibodies: Screening for Anti-HABA Antibodies with DifferentSpecificities.

Purity of the affinity purified anti-HABA polyclonal antibodiesaccording to Example 7.5 above was verified by SDS gel electrophoresis.Concentration of an affinity purified anti-HABA antibody solution wasdetermined spectrophotometrically using the average ε^(%) _(280 nm)value of 14.5 for IgGs. Specificity of anti-sera and affinity purifiedantibodies for different epitopes in the HABA molecule was verified byELISA and UV spectrophotometry.

7.7 ELISA Assay

Ninety-six well microtiter plates (Nunc F96, Maxisorp) were coated byovernight incubation at 4° C. with 50 μl/well of HABAylated avidinsolution (10 μg/ml in 0.05 M Na carbonate, pH 9.5). Plates were washedthree times with PBS/Tween 0.05% (PBS/T) and blocked by adding 200 μl ofPBS/T containing 3% BSA or 0.1% of gelatine. After 2 hours incubation at37° C., plates were washed three times with PBS/T.

Serial dilutions of antisera or affinity purified anti-HABA antibodies(50 μl) were then incubated for 2 h at 37° C. When HABAylated avidin wasused for the coating, the experiment was run in duplicate and theantibodies were incubated with and without biotin in the dilutingbuffer. Plates were washed 3 times with PBS/T and incubated for 2 hoursat 37° C. with 50 μl/well of a solution containing HRP-conjugatedanti-rabbit antibodies (diluted 1:2,500). After extensive washing withPBS/T, 100 μl of o-phenylenediamine solution were added, the reactionstopped using 1M H₂SO₄ and the OD at 490 nm was measured after 5minutes.

Absence of cross reactivity against the anthranilic part of the HABAmolecule was verified running a control ELISA assay with BSA-anthranilicacid in the first coating. As shown in FIG. 3, anti-HABA antibodiespurified on GEL A were able to recognize HABA as part of the HABAylatedavidin in the absence (⋄) as well as in the presence of biotin (♦).However, in the absence of biotin, the anti-HABA antibody purified onGEL B failed to recognize the HABA buried in the binding site. Uponaddition of biotin, however, the HABA moiety was expelled and strongbinding of the anti-HABA antibody was detected (

). This effect clearly depends on the procedure used for purification ofthe anti-HABA antibodies.

7.8 Spectrophotometry

UV spectra of HABA (compound 0) in PBS was recorded in the presence ofthe anti-HABA antibodies affinity purified in both GELs A and B. Theresults indicate that:

Antibodies purified with GEL A (A-anti-HABAs) recognize the HABA moietywhen it is either in the azo or the quinone conformation.

Antibodies purified with GEL B (B-anti-HABAs) can bind to the HABAmoiety only when it is in the azo conformation whereas they fail torecognize it in the quinone conformation. In this case, recognition ofthe HABAylated avidin occurs only after biotin expels HABA from thebinding pocket.

EXAMPLE 8

Single-Layer Protein System

To prove this principle, HABA was coupled covalently to the binding siteof avidin using an appropriate spacer arm. The introduction of biotin orbiotinylated compounds served to expel the HABA moiety from the bindingsite, thereby rendering it available for further interaction with otherbinding molecules, e.g., unmodified avidin, anti-HABA antibodies orbiotinylated anti-HABA antibodies (FIG. 1). This self-contained systemcan be used to generate growing cascades of interactions and layers.

As a second HABA-binding molecule; anti-HABA polyclonal antibodiesaffinity purified on GEL B were used to selectively built a single layersystem that is dependent on the addition of biotin as a trigger (FIG.3), showing that the HABA moiety is indeed buried in the avidin bindingsite and is not available for further interaction before itsdisplacement by biotin.

EXAMPLE 9

Multilayer Protein System

To prove that this system meets the requirements of a signaltransduction cascade and the assemblage of protein multilayers (Mülleret al, 1993), biotin-saturated HABAylated avidin was incubated withbiotinylated anti-HABA antibodies, followed by additional cycles ofHABAylated avidin and biotinylated anti-HABA antibodies. A stepwiseincrease in absorbance could be detected after the formation of eachlayer (FIG. 4). The assemblage of multilayers could be initiated usingHABAylated avidin and either biotin or biotinylated macromolecules. Ineither case, the addition of biotin was crucial to expel the HABA fromthe binding site, thus enabling subsequent interaction with theanti-HABA antibodies.

Since avidin has 4 binding sites, if limited amounts of biotin are addedto displace only one or two HABA molecules, the system can be triggeredvectorially in different dimensions.

APPENDIX A

APPENDIX B Structures of Compounds 1–11

REFERENCES

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Bayer, E. A.; Wilchek, M. (1990), Methods Enzymol. 184, 138.

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1. A single-layer protein system, comprising: (i) a protein being anavidin-type molecule selected from the group comprising native egg-whiteavidin, recombinant avidin, deglycosylated avidins, bacterialstreptavidin, recombinant streptavidin, truncated streptavidin and otherderivatives of said avidin-type molecules; (ii) two ligands I and IIwhich bind with different affinities at the same binding site of saidprotein, wherein said ligand I is 4′-hydroxyazobenzone-2-carboxylic acid(HABA) or a HABA derivative, which is the low affinity ligand, and saidligand II is biotin, which is the high affinity ligand; and (iii) amolecule that recognizes the low affinity ligand I, wherein saidmolecule is an anti-HABA antibody or a biotinylated anti-HABA antibody,wherein in said single-layer protein system the high affinity ligand IIis buried within the binding site of the protein (i) and the lowaffinity ligand I is covalently bound to the protein and associated withthe molecule (iii) that recognizes it.
 2. A single-layer protein systemaccording to claim 1, wherein the molecule (iii) is labeled with highaffinity ligand II.
 3. A multilayer protein system comprising two ormore layers according to claim
 2. 4. A multilayer protein systemaccording to claim 3 wherein in the last layer molecule (iii) is notlabeled with high affinity ligand II.
 5. A multilayer protein systemaccording to claim 3, formed on a substrate selected from the groupconsisting of gold, silicium, polystyrene.
 6. A multilayer proteinsystem according to claim 3 comprising 5–6 layers.
 7. A method forassembling a multilayer protein system according to claim 3, whichcomprises the steps of: (a) covalently binding said low affinity ligandI to said protein (i), thus obtaining a low affinity ligand I-protein(i) complex in which said ligand I is buried within the binding site ofsaid protein (i) and is thus not available for interaction with othermolecules that recognize it; (b) reacting the high affinity ligand II ora compound containing said high affinity ligand II with the low affinityligand I-protein (i) complex of step (a) above, whereby low affinityligand I is expelled from within the binding site to the periphery butremains covalently bound to protein (i) and high affinity ligand II isassociated to, and buried within, the binding site of protein (i); (c)reacting the low affinity ligand I-protein (i)-high affinity ligand IIcomplex of step (b) with said molecule (iii) that recognizes and bindsto low affinity ligand I and is labeled with high affinity ligand II;and (d) reacting the protein system of step (c) with low affinity ligandI-protein (i) complex as in step (b) above, and repeating steps (c) and(d) as desired.
 8. A single-layer protein system according to claim 1,formed on a substrate selected from the group consisting of gold,silicium, polystyrene.
 9. A method for assembling a single-layer proteinsystem according to claim 1, which comprises the steps of: (a)covalently binding said low affinity ligand I to said protein (i), thusobtaining a low affinity ligand I-protein (i) complex in which saidligand I is buried within the binding site of said protein (i) and isthus not available for interaction with other molecules that recognizeit; (b) reacting the high affinity ligand II or a compound containingsaid high affinity ligand II with the low affinity ligand I-protein (i)complex of step (a) above, whereby low affinity ligand I is expelledfrom within the binding site to the periphery but remains covalentlybound to protein (i) and high affinity ligand II is associated to, andburied within, the binding site of protein (i); and (c) reacting the lowaffinity ligand I-protein (i)-high affinity ligand II complex of step(b) with a molecule (iii) that recognizes and binds to low affinityligand I.